3 La complexité du trouble dépression majeure (MDD)PURPOSE OF THE SLIDETo transition to the first section of the presentation, which will be to discuss the complexity of major depressive disorder (MDD) and conceptualizing it beyond the core mood symptoms.

4 Origines multiples et symptômes associésSymptomes émotionelsCulpabilitéSuicidePerte d’intérêtsTristesseSymptomes associésRegressionRuminations obsessionellesIrritabilitéPréoccupations somatiques excessivesDouleursLes pleursAnxiété et phobiesSymptomes physiquesManque d’énergieDiminution de la concentrationTroubles alimentaireTroubles du sommeilTroubles psychomoteursPURPOSE OF THE SLIDEEstablish that depression is a complex disorder that can be manifested through a variety of emotional, physical, and other associated symptoms (e.g., anxiety, worry, and pain).Note that the complexity of the symptom presentation can lead to depression being a difficult condition to diagnose.The variety of symptoms of depression suggests that many areas of the brain and neural networks may be involved in depression.KEY POINTSThere is a broad range of major depressive disorder (MDD) symptoms.The mood in major depression is a period of at least 2 weeks during which there is either depressed mood or the loss of interest or pleasure in nearly all activities. The mood is often described by the person as depressed, sad, hopeless, discouraged, or “down in the dumps”.Some individuals emphasize somatic complaints (e.g., body aches and pains) rather than reporting feelings of sadness. Many individuals report or exhibit increased irritability. Loss of interest or pleasure is nearly always present, at least to some degree, and not feeling enjoyment in activities that were previously considered pleasurable.Appetite is usually reduced, yet other individuals have increased appetite. When appetite changes are severe, there may be significant loss or gain in weight.The most common sleep disturbance associated with depression is insomnia. Less frequently, individuals present with over-sleeping in the form of prolonged sleep episodes at night or increased daytime sleep.Psychomotor changes include agitation (e.g., the inability to sit still, pacing, hand-wringing) or retardation (e.g., slowed speech, thinking, and body movements). Decreased energy, tiredness, and fatigue are common.The sense of worthlessness or guilt associated with depression may include unrealistic negative evaluation of one's worth or guilty preoccupations or ruminations over minor past failings.Many individuals report impaired ability to think, concentrate, or make decisions. They may appear easily distracted or complain of memory difficulties.Frequently, there may be thoughts of death, suicidal ideation, or suicide attempts.BACKGROUNDThe symptom groupings were based on the DSM-IV-TR core criteria and text description of associated symptoms.REFERENCEAmerican Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. Text Revision. 4th ed. Washington, DC: American Psychiatric Association; 2000;352,356.American Psychiatric Association (APA). DSM-IV-TR; 2000:352,356.

7 Evolution Progressive et Chronique de la Dépression majeures (MDD)PURPOSE OF THE SLIDETo transition to the next section of the presentation regarding the course of major depressive disorder (MDD).KEY POINTSMDD is believed to be an episodic illness, but for most patients with MDD, it is experienced recurrently.Research findings suggest that recurrent and chronic MDD may be associated with underlying neurobiological alterations.

8 Progression de la Dépression -“Kindling”- Effets de chaque épisode Successif102468Likelihood of recent life stress precipitating depressionRisk (odds ratio) of depression onset per monthFemale participants only N=2,395Risk (odds ratio)PURPOSE OF THE SLIDETo discuss that the progressive nature of major depressive disorder (MDD) is shown by the relationship between onset, life events, and the previous number of depressive episodes.KEY POINTSMembers of female–female twin pairs from a population-based registry (N=2,395), who were interviewed four times over a period of 9 years, formed a study group that contained 97,515 person–months and 1,380 onsets of major depression.Discrete-time survival, proportional hazards model, and piece-wise regression analyses were used to examine the interaction between life event exposure and number of previous depressive episodes in the prediction of episodes of major depression.For those with zero to nine previous depressive episodes, the depressogenic effect of stressful life events declined substantially with increasing episode number.However, the association between stressful life events and major depression was not substantially influenced by additional episodes.This pattern of results was robust to the addition of indices of event severity, measures of genetic risk, and restriction to independent stressful life events. The same pattern was also seen upon examining within-person changes in number of episodes.Thus, these results are consistent with the kindling hypothesis but suggest a threshold at which the mind/brain is no longer additionally sensitized to the depressive state. Chief predictor of future recurrence after nine episodes is the episode count, not presence of stress.REFERENCEKendler KS, et al. Stressful life events and previous episodes in the etiology of major depression in women: an evaluation of the "kindling" hypothesis. Am J Psychiatry. 2000;157(8):1243–1251.1234567–89–11Nombre d’épisode dépressifs antérieursKendler KS, et al. Am J Psychiatry. 2000;157:1243–1251.

9 Les Symptômes physiques ne répondent pas de façon équivalente aux traitements antidépresseurs1.4Emotional symptomsN=573Non-somatic depressive symptoms1.2Positive well-being1.0Physical symptoms0.8ImprovementNon-pain somatic symptomsTreatment effect size0.6Pain somatic symptoms0.40.2PURPOSE OF THE SLIDETo point out that painful physical symptoms are often residual symptoms, even with treatment, and thus patients can continue to be at risk for recurrence.KEY POINTSA naturalistic, randomized trial of 573 primary care patients taking selective serotonin reuptake inhibitors (SSRIs) showed that, over time, emotional and physical symptoms of major depressive disorder (MDD) respond differently to treatment.Adult patients considered clinically depressed by their primary care physician were randomized to SSRI treatment from 37 practice sites.Depression outcome was assessed by the 9-item Patient Health Questionnaire (PHQ-9) depression scale and The Hopkins Symptoms Checklist subscale (HSCL-20). Physical symptoms were assessed by 14 of the 15 items on the PHQ-15 (sexual dysfunction was excluded). Social, work and well being parameters were determined using the 36-item Short-form Health Survey (SF-36), the Work Limitations Questionnaire, and the Medical Outcomes Study.Following treatment with an antidepressant, mood symptoms of MDD improved; however, physical symptoms, and, in particular, painful physical symptoms, were more likely to persist.BACKGROUNDTreatment-effect size: this analysis provides a way to make comparisons that adjust for the variability in outcomes. It is calculated by dividing the difference (mean change) by the standard deviation.REFERENCEGreco T, et al. The outcome of physical symptoms with treatment of depression. J Gen Intern Med. 2004;19:813–818.Baseline1 month3 months6 months9 monthsARTIST=A Randomized Trial Investigating SSRI Treatment; SSRI=selective serotonin reuptake inhibitor.Adapted from: Greco T, et al. J Gen Intern Med. 2004;19:813–818.

10 Les Symptômes douloureux résiduels diminuent la rémission dans la DépressionÉtude longitudinale de 3 ans, adultes agés 55–851020304050607080MDD onlyMDD and painfulsymptoms% Patients (depressed at baseline)achieving endpoint recoveryN=102N=1199%47%(PURPOSE OF THE SLIDETo reinforce the impact of painful physical symptoms on the course and recovery from depression.KEY POINTSThe study was part of the Longitudinal Aging Study Amsterdam (LASA), which drew a random sampling of 3,107 adults aged 55–85.Data were collected at 5-month intervals over 3 years. Two consecutive measurements of pain or depression were necessary to contribute to the analyses.The Center for Epidemiologic Studies Depression scale (CES-D), used commonly in the EU, is a 20-item self-reported scale measuring depressive symptoms. Items are scored on how often subjects experience each symptom in the previous week: 0 (rarely or none of the time) to 3 (most or all of the time). A score of ≥16 (out of 60) is a general measure of clinically relevant depressive symptoms.Depressed = CES-D ≥16; Recovery from depression = CES-D <16.Pain was determined by a subscale of the Nottingham Health Profile (NHP).Patients answered yes or no to the following: I am in pain when I am standing; I find it painful to change position; I am in pain when I am sitting; I am in pain when I walk; I am in constant pain.Total scores ranged from 0 (low) to 5 (high). Subjects with “pain” were those with a Nottingham score ≥1. Recovery from pain = 0 (low or no pain) on the NHP.When painful symptoms and depression coexist, chances of recovery from depression are greatly diminished.In this study, nearly half of those patients diagnosed with depression recovered by endpoint. This compares with a recovery rate of just 9% for those who experienced both depression and pain at baseline.REFERENCEGeerlings SW, et al. Longitudinal relationship between pain and depression in older adults: sex, age and physical disability. Soc Psychiatry Psychiatr Epidemiol. 2002;37:23–30.La relation étroite entre symptômes douloureux and dépression démontre la nécessité de stratégies de traitements progressivesMDD=major depressive disorder.Geerlings SW, et al. Soc Psychiatry Psychiatr Epidemiol. 2002;37:23–30.

11 Modifications Fonctionnelles et Structurales de cerveau dans la MDD et la DouleurPURPOSE OF THE SLIDETo transition to the next section of the presentation.KEY POINTSAt this point, we have discussed the complexity of the presentation of major depressive disorder (MDD), that it often has a recurrent and chronic course, and that it may be progressive over time with greater risk for recurrence related to residual symptoms, such as painful physical symptoms, and recurrent episodes.In the following section we focus on some of the underlying neurobiologic changes associated with the occurrence of MDD.

14 Orbitofrontal cortical (gyrus rectus)Les Patients avec MDD présentent un cortex Orbitofrontal médian plus petit que les Contrôles*P=.02Orbitofrontal cortical (gyrus rectus)volume (mm3)MOFC*PURPOSE OF THE SLIDETo look at some of the potential structural changes observed in patients with depression.Reduced cortical volumes have been found in subregions of the frontal cortex, including the subgenual anterior cingulate and medial orbitofrontal cortex.KEY POINTSNeuroimaging studies have begun to show how functional differences in the brain between depressed and healthy patients can be associated with structural differences—though we still don’t know how this happens.The medial orbitofrontal cortex (MOFC; shaded in red on the image at left) has a role in the regulation of emotion and mood. In patients with depression (N=15), the MOFC was 32% smaller in volume than controls (N=20), as is indicated in the chart on the right. This finding was significant after statistically controlling for brain size. The authors hypothesize that decreases in volume of the MOFC in patients may be a risk factor for the development of depression.BACKGROUNDThe study consisted of 15 patients with a history of DSM-IV depression and currently treated with antidepressant medication (paroxetine, fluoxetine, or desipramine). Patients with a history of post-traumatic stress disorder (PTSD) or current medication use other than antidepressants were excluded.Comparison subjects (N=20) were healthy subjects selected to be similar to the patients for gender, age, years of education, and handedness.Depressed patients, on average, were in remission for 30 weeks and had an average of two prior depressive episodes.REFERENCEBremner JD, et al. Reduced volume of orbitofrontal cortex in major depression. Biol Psychiatry. 2002;51:273–279.Patients avec MDD ont une réduction de 32% MOFC (VMPFC) que les patients contrôlesMOFC=medial orbitofrontal cortices; VMPFC=ventromedial prefrontal cortex.Bremner JD, et al. Biol Psychiatry. 2002;51:273–279.

16 La douleur chronique peut entraîner une atrophie de la matière grise cérébraleLes zones en rouge Indiquent les régions où la densité de matière grise est réduite chez les patients (CBP) par rapport aux contrôlesUne tranche du thalamus antérieur droit à la pointe de la diminution de la matière grise thalamiquePURPOSE OF THE SLIDETransition to structural changes that may be observed in gray matter in patients with chronic pain conditions.KEY POINTSThis is the first study showing brain structural changes in patients with chronic pain.26 patients fulfilling the International Association for the Study of Pain (IASP) criteria for chronic back pain (CBP) with pain duration of at least 1 year were compared with 26 matched volunteers.The source of CBP was not distinguished, although the main source of pain was lumbosacral.Voxel-based morphometry (VBM) has been validated against other standard measures. Images were first normalized into a standard space and then segmented. Voxel values reflect absolute amounts (volume in arbitrary units) of gray matter.In the VBM image on the left, gray matter density is bilaterally reduced in the dorsolateral prefrontal cortex (PFC). These observed changes likely constitute the cognitive and behavioral properties (pain perception) of chronic pain.The image on the right shows a significant decrease in gray matter density was observed in the right anterior thalamus. The left thalamus also showed a decrease in gray matter density; however, this decrease did not pass the significant threshold. The thalamus has an importance in mediating nociceptive inputs to the cortex.All told, patients with CBP had 5–11% less neocortical gray matter volume, similar to the effects of 10 to 20 years of normal aging compared with controls. The decrease in volume was related to pain duration, indicating a 1.3 cm3 loss of gray matter for every year of chronic pain.These are meaningful differences, though association cannot explain causality.The authors hypothesize that as atrophy of elements of the circuitry progresses, the pain condition becomes more irreversible and less responsive to therapy.REFERENCEApkarian AV, et al. Chronic back pain is associated with decreased prefrontal and thalamic gray matter density. J Neurosci. 2004;24(46):10410–10415.Les patients atteints de CBP ont 5-11% de matière grise en moins que la population normale, ce qui équivaut à ans de vieillissement normal.Images copyright 2006 by the Society for NeuroscienceApkarian AV, et al. J Neurosci. 2004;24:10410–10415.

17 La Dépression et la Douleur interfèrent avec les voies de communications intracérébraleMDDCognitive/executivecortexDorsolateral PFCDorsal ACCDouleur?Limbic formationsHippocampusAmygdalaNucleus accumbensIntegrative cortexLateral orbital PFCRostral PFCMedial PFCEmotional/visceralcortexVentral ACCVentral medial PFCHypothalamusPURPOSE OF THE SLIDETo summarize in general how depression impacts cortico–limbic circuitry.KEY POINTSHere we see a simplified graphical representation of the cortico–limbic feedback loop.If there is activation in the limbic part, this activation will lead to increased activity in the paralimbic prefrontal cortex that will then go towards integrative cortex to the executive function cortex, so it is emotion, behavior, cognition, and then shutting down the limbic system.In people who have depression, the loop never seems to close in an adaptive way. Emotions have lost their adaptive value. There is increased activity in limbic formation that persists and that drives hypothalamus and causes increased sympathetic activity that causes increase in the hypothalamic pituitary adrenal (HPA) axis.Something that has started in the mind has now propagated to the rest of the body.Pain, too, may inhibit cortico–limbic feedback, but to what degree and how is the subject of debate.1It is these three circles on the bottom right that we have focused most of our attention on.BACKGROUNDAt least one study suggests that the processing of pain in limbic formations such as the hippocampus and entorhinal cortex affects connectivity with the dorsolateral prefrontal cortex (PFC) and therefore impacts the perception of chronic pain.1Patients with functional illnesses such as depression may have altered pain processing. The mechanisms underlying altered pain processing in depression are unknown, but may include changes in the HPA axis. More subtle changes in neurotransmitter systems and other forms of neural plasticity may also contribute.2REFERENCESCasey KL, Tran ATD. Cortical mechanisms mediating acute and chronic pain in humans. In: Handbook of Clinical Neurology. 3rd series. Boston, MA: Elsevier;2006:159–77.Borsook D, et al. Reward-aversion circuitry in analgesia and pain: Implications for psychiatric disorders. Eur J Pain. 2007;11:7–20.Illustration based on:Mayberg HS. Positron emission tomography imaging in depression: a neural systems perspective. Neuroimaging Clin N Am. 2003;13:805–815.Drevets WC. Neuroimaging and neuropathologic studies of depression: implications for the cognitive-emotional features of mood disorders. Curr Opin Neurobiol. 2001;11:240–249.Phillips ML, et al. Neurobiology of emotion perception II: Implications for major psychiatric disorders. Biol Psychiatry. 2003;54:515–528.Ongur D, Price JL. The organization of networks within the orbital and medial prefrontal cortex of rats, monkeys and humans. Cerebral Cortex. 2000;10:206–219.Phan KL, et al. Functional neuroanatomy of emotion: a meta-analysis of emotion activation studies in PET and fMRI. Neuroimage. 2002;16:331–348.Siegle CJ, et al. Increased amygdala and decreased dorsolateral prefrontal BOLD responses in unipolar depression: related and independent features. Biol Psychiatry. 2007;61:198–209.Tracey I. Nociceptive processing in the human brain. Curr Opin Neurobiol. 2005;15:478–487.Hariri AR, et al. Imaging genetics: perspectives from studies of genetically driven variation in serotonin function and corticolimbic affective processing. Biol Psychiatry. 2006,59:888–897.Brody AL, et al. Brain metabolic changes associated with symptom factor improvement in major depressive disorder. Biol Psychiatry. 2001;50:171–178.ACC=anterior cingulate cortex; MDD=major depressive disorder; PFC=prefrontal cortex.Maletic V, et al. Int J Clin Pract. 2007;61:2030–2040.

19 Similitudes des troubles entre l’axe Hypothalamo-cortico-surrénalien et les CytokinesStress and Depression1,2Pain3PURPOSE OF THE SLIDETo introduce neuroendocrine and neuroimmune changes associated with depression, pain, and stress.KEY POINTSMolecular and cellular studies of stress, depression, and antidepressants have moved the field of mood disorder research beyond the monoamine hypothesis of depression. These studies demonstrate that stress and antidepressant treatment exert opposing actions on the expression of specific neurotrophic factors in limbic brain regions involved in the regulation of mood and cognition.1,2Stress and depression:A mechanism by which the brain reacts to stress and depression is activation of the hypothalamus–pituitary–adrenal (HPA) axis. The activity of the HPA axis is controlled by several brain pathways, including the hippocampus (which exerts an inhibitory influence on hypothalamic corticotropin-releasing factor (CRF) containing neurons) and the amygdala (which exerts a direct excitatory influence on hypothalamic CRF containing neurons). Levels of glucocorticoids that are seen under normal physiological circumstances seem to enhance hippocampal inhibition of HPA activity and may enhance hippocampal function and therefore promote cognitive abilities. However, under sustained elevations of glucocorticoids, seen under conditions of prolonged, severe stress and possibly in patients with depression, hippocampal neurons may be damaged and reduction of neurogenesis may occur. Abnormal, excessive activation of the HPA axis has been observed in approximately half of individuals with depression.3Patients with major depression who are otherwise medically healthy have also been observed to have activated inflammatory pathways. Activation of macrophages due to inflammatory challenges (infection, tissue damage, or destruction) may cause release of proinflammatory cytokines. These cytokines enter several areas of the afferent sensory systems and can lead to increased activity. Once in the brain, cytokines can cause altered metabolism of serotonin (5-HT) and dopamine (DA), activation of corticotrophin-releasing hormone (CRH) leading to increased serum glucocorticoid levels, and disruption of synaptic plasticity through changes in growth factors such as brain-derived neurotrophic factor (BDNF).4Pain:5Similarly, chronic stress evoked by chronic pain can lead to loss of negative glucocorticoid feedback on the HPA axis, driving this axis to generate more glucocorticoids by downregulation of the glucocorticoid receptor within the brain and periphery. Inflammation and nerve injury stimulate neurons in the dorsal horn of the spinal cord, which then relay pain signals to the thalamus.Monoamines in the brainstem normally descend to the spinal cord to act as a “brake” on nociceptive transmission. Increased glucocorticoid activity during chronic pain can lead to monoamine depletion in descending inhibitory systems. Loss of glucocorticoid inhibition of proinflammatory cytokines leads to increased peripheral inflammation, contributing to pain sensitization.Although acute stress is analgesic, chronic stress from chronic pain can lead to downregulation of inhibitory circuits between the limbic system and somatosensory cortex, leading to enhanced pain perception. Chronic pain-induced downregulation of inhibitory limbic-somatosensory circuits may also lead to depressed mood.REFERENCESDuman R. Depression: a case of neuronal life and death? Biol Psychiatry. 2004;56:140–145.Duman R. Role of neurotrophic factors in the etiology and treatment of mood disorders. Neuromolecular Med. 2004;5:11–25.Nestler EJ, et al. Neurobiology of depression. Neuron. 2002;34(1):13–25.Raison CL, et al. Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends in Immunol. 2006;27:24–31.Blackburn-Munro G, et al. Chronic pain, chronic stress and depression: coincidence or consequence? J Neuroendocrinol. 2001;13:1009–1023.red=inhibitory pathways to hypothalamus–pituitary–adrenal (HPA) axis; green=stimulatory pathways to HPA axisAdapted from: 1. Raison, et al. Trends in Immunol. 2006;27:24– Nestler EJ, et al. Neuron. 2002;34:13– Blackburn-Munro G, et al. J Neuroendocrinol. 2001;13:1009–1023.

28 Rôle du traitement antidépresseur dans la restoration neurobiologiquePURPOSE OF THE SLIDEAt this point, the underlying structural, functional, neuroendocrine, neuroimmune, and cellular alterations have been reviewed.What is the evidence that antidepressant treatment can restore these systems?

29 Voie nociceptive ascendante5HT et Norepinephrine modulateurs de la perception de l’humeur et de la douleur1–3Locuscoeruleus(source de NE)Raphe nuclei(source de 5-HT)AmygdalaHippocampeVoie NE descendanteVoie 5HT descendanteVoie nociceptive ascendanteSystème limbiqueCortex prefrontalKEY POINTSOne of the guiding principles in the development of pharmacotherapeutics for depression has been the monoamine hypothesis.1While some findings have not been universally confirmed, there is considerable support for dysfunctional serotonergic or noradrenergic neurotransmission in patients with depression.1Serotonin (5-HT) and norepinephrine (NE) are part of the body's endogenous analgesic system, which inhibits transmission of ascending pain signals from the periphery.2Because 5-HT and NE are key neurotransmitters in both mood regulation and in the modulation of pain perception, it is hypothesized that increasing the activity of both 5-HT and NE may help in the treatment of mood disorders as well as in pain disorders.BACKGROUND5-HT and NE are also involved in directly modulating pain at the level of the dorsal horn in the spinal cord.5-HT both inhibits and facilitates the perception of pain. 5-HT inhibits pain via the descending inhibitory arm of the descending modulatory pathway, and it facilitates the perception of pain via the descending facilitatory arm of the descending modulatory pathway.2,3NE inhibits the perception of pain via the descending inhibitory arm of the descending modulatory pathway. NE does not seem to be involved in the facilitatory aspect of pain perception in the descending modulatory pathway.2REFERENCESBymaster FP, et al. The dual transporter inhibitor duloxetine: a review of its preclinical pharmacology, pharmacokinetic profile, and clinical results in depression. Curr Pharm Des. 2005;11(12):1475–1493.Fields HL, et al. Neurotransmitters in nociceptive modulatory circuits. Annu Rev Neurosci. 1991;14:219–245.Fields H. State-dependent opioid control of pain. Nat Rev Neurosci. 2004;5(7):565–575.5-HT=serotonin; NE=norepinephrine.Adapted from: 1. Bymaster FP, et al. Curr Pharm Des. 2005;11:1475– Fields H. Nat Rev Neurosci. 2004;5:565– Fields HL, et al. Annu Rev Neurosci. 1991;14:219–245.

38 L’amélioration des symptômes douloureux dans la MDD augmente les chances de rémission100*P<.0012 pooled studies*36%Patients en rémission (études de 9-semaines) (%)18%N=77N=4950% Amélioration des symptômesPhysiques douloureux50% Amélioration des symptômesphysiques douloureuxPURPOSE OF THE SLIDENot only is pain common among patients with depression, but improvement of pain symptoms associated with depression may improve chances for remission.Successful treatment of pain symptoms may be an important consideration for optimizing depression treatment outcomes.KEY POINTSPain symptoms associated with depression may have an important impact on depression outcomes.In the two 9-week studies by Fava, successful treatment of pain was associated with a significantly greater chance of achieving remission.Note: The two groups (of ≥50% or <50% improvement in painful physical symptoms) were not divided based on treatment given, so both groups could contain patients randomized to either active treatment or placebo.These findings underscore the urgency to address painful symptoms along with other prominent symptoms of depression.BACKGROUNDIn the Fava study, pooled data from two different studies where patients were randomized to receive active treatment (N=244) or placebo (N=251) revealed a similar relationship in the remission rate.The remission rate for pain responders (50% improvement in Visual Analog Scale [VAS] overall pain) was twice that observed for pain nonresponders (36.2% versus 17.8%, P<.001).Of 495 patients with depression:Remission was defined as a Hamilton Depression Rating Scale (HAMD)-17 total score 7.Painful physical symptom improvement was measured by VAS for overall pain from baseline to last observation of 50% or greater.Patients showing a pain response within the first two weeks of starting therapy had an estimated 35.4% probability of achieving depressive symptom remission versus 20.9% for patients not showing early pain response.REFERENCEFava M, et al. The effect of duloxetine on painful physical symptoms in depressed patients: do improvements in these symptoms result in higher remission rates? J Clin Psychiatry. 2004;65(4):521–530.Rémission=HAMD-17 total score 7Fava M, et al. J Clin Psychiatry. 2004;65:521–530.